Maturation, desiccation and encapsulation of gymnosperm somatic embryos

Abstract

PCT No. PCT/CA92/00549 Sec. 371 Date Aug. 18, 1994 Sec. 102(e) Date Aug. 18, 1994 PCT Filed Dec. 18, 1992 PCT Pub. No. WO93/11660 PCT Pub. Date Jun. 24, 1993A desiccated mature gymnosperm somatic embryo characterized by having a moisture content which is lower than the moisture content of its corresponding zygotic embryo and a dry weight and per embryo lipid content which are higher than the lipid content and dry weight of its corresponding gymnosperm zygotic embryo. Preferred desiccated gymnosperm somatic embryos are conifer somatic embryos having a moisture content ranging between 10 and 55%. Also within the scope of the invention is a method for producing desiccated somatic embryos. The method comprises developing immature embryos in a medium comprising at least one non-permeating water stress agent, a metabolizable carbon source and a growth regulator having an influence on embryo development for a period of time sufficient to yield the recovery of mature somatic embryos having a moisture content which is lower than the moisture content of corresponding zygotic embryos. The method can optionally include a secondary desiccation treatment to further reduce the moisture content of the embryos.

We claim: 
     
       1. A method for producing viable desiccation-tolerant gymnosperm somatic embryos, the method comprising developing immature gymnosperm somatic embryos in the presence of at least one water stress agent, a metabolizable carbon source, and a growth regulator having an influence on embryo development, for a period of time sufficient to obtain gymnosperm somatic embryos tolerant of desiccation to a moisture content of less than 55%. 
     
     
       2. The method according to claim 1, comprising additionally desiccating the desiccation-tolerant somatic embryos in the presence of a water stress agent to a moisture content of less than 55%. 
     
     
       3. The method according to claim 2, wherein said gymnosperm is a conifer. 
     
     
       4. A method for producing mature desiccation-tolerant conifer somatic embryos, said method comprising developing immature conifer somatic embryos in presence of at least one osmotic stress agent, a metabolizable carbon source, and a growth regulator having an influence on embryo development, for a period of time sufficient to obtain mature desiccation-tolerant somatic embryos having a moisture content of from about 32 to 55%. 
     
     
       5. A method for producing viable desiccated conifer somatic embryos, said method comprising (1) developing immature conifer somatic embryos in presence of at least one osmotic stress agent, a metabolizable carbon source, and a growth regulator having an influence on embryo development, for a period of time sufficient to obtain mature desiccation-tolerant somatic embryos having a moisture content of from about 32 to 55%, and (2) submitting said mature somatic embryos to a secondary desiccation treatment to further reduce the moisture content of said embryos. 
     
     
       6. A method according to claim 4 or claim 5 wherein said osmotic stress agent is a metabolizable carbon source. 
     
     
       7. A method according to claim 4 or claim 5 wherein said osmotic stress agent is substantially non-permeating. 
     
     
       8. A method according to claim 4 or claim 5, wherein said growth regulator is a stress hormone. 
     
     
       9. A method according to claim 4 or claim 5, wherein said growth regulator is selected from abscisic acid and/or analogs, precursors or derivatives thereof. 
     
     
       10. A method according to claim 9, wherein said abscisic acid and/or analogs, precursors or derivatives thereof are initially present in sufficient concentration to have a final concentration of abscisic acid and/or analogs, precursors or derivatives thereof of at least 0.1 μM at the end of the development of said mature somatic embryos. 
     
     
       11. A method according to claim 9, wherein said analogs, precursors or derivatives of abscisic acid are selected from the group consisting of abscisyl alcohol, acetylenic aldehyde, dihydroacetylenic alcohol, phaseic acid (PA), dihydrophaseic acid (DPA), 6'-hydroxymethyl abscisic acid (HM-ABA), beta-hydroxy abscisic acid, beta-methylglutaryl abscisic acid, beta-hydroxybeta-methylglutarylhydroxy abscisic acid, 4'-desoxy abscisic acid, abscisic acid beta-D-glucose ester 2-2(2-p-chlorophenyltrans-ethyl)cyclopropane carboxylic acid and jasmonic acid and functional derivatives thereof. 
     
     
       12. A method according to claim 9, wherein the concentration of abscisic acid ranges from 0.1 to 100 μM. 
     
     
       13. A method according to claim 4 or claim 5, wherein said immature embryos are developed in step (1) for a period of time sufficient to obtain mature desiccation-tolerant conifer somatic embryos having a moisture content ranging between substantially 32 and 55%. 
     
     
       14. A method according to claim 13, wherein the mature desiccation-tolerant somatic embryos have a moisture content ranging between substantially 32 and 45%. 
     
     
       15. A method according to claim 4 or claim 5, further comprising a preliminary step which comprises preculturing said immature embryos in presence of a reduced auxin cytokine medium or a reduced auxin medium to promote maturation of said embryos. 
     
     
       16. A method according to claim 4 or claim 5, wherein said immature embryos are developed in a bioreactor vessel. 
     
     
       17. A method according to claim 16, wherein said vessel is a continuous-flow solid-support bioreactor vessel. 
     
     
       18. A method according to claim 4 or claim 5, wherein a substantially changing concentration of said growth regulator is supplied during developing of said immature embryos. 
     
     
       19. A method according to claim 4 or claim 5, wherein a substantially constant concentration of said growth regulator is supplied during developing of said immature embryos. 
     
     
       20. A method according to claim 4 or 5, wherein said embryos are from the family Pinaceae. 
     
     
       21. A method according to claim 4 or claim 5, wherein said immature embryos are developed for a period of time ranging from 1 to 15 weeks. 
     
     
       22. A method according to claim 4 or claim 5, wherein said osmotic stress agent is present in a sufficient amount to maintain an osmotic potential ranging between -0.3 and -2.0 MPa. 
     
     
       23. A method according to claim 4 or claim 5, wherein a substantially changing concentration of said osmotic stress agent is supplied during developing of said immature embryos. 
     
     
       24. A method according to claim 4 or claim 5, wherein a substantially constant concentration of said osmotic stress agent is supplied during developing of said immature embryos. 
     
     
       25. A method according to claim 4 or claim 5, wherein said osmotic stress agent is a compound having a molecular size of at least 30 Ångstroms. 
     
     
       26. A method according to claim 25, wherein said osmotic stress agent is polyethylene glycol. 
     
     
       27. A method according to claim 26, wherein said polyethylene glycol is present at a concentration ranging between 1 and 30% wt. 
     
     
       28. A method according to claim 5, wherein step (2) comprises submitting said mature somatic embryos to at least one relative humidity environment. 
     
     
       29. A method according to claim 28, wherein said mature embryos are further desiccated by being submitted to one or a plurality of environments having a relative humidity ranging between 5 and 100%. 
     
     
       30. A method according to claim 29, wherein said mature embryos are, prior to storage, submitted to said environment of relative humidity for a period of time ranging from 24 to 48 hours to achieve moisture loss. 
     
     
       31. A method according to claim 5, wherein step (2) comprises submitting said mature somatic embryos to a further osmotic stress agent. 
     
     
       32. A method according to claim 31, wherein the further osmotic stress agent is a substantially non-permeating water stress agent. 
     
     
       33. A method according to claim 5, wherein after step (2), said desiccated somatic embryos have a moisture content below about 36%. 
     
     
       34. A method according to claim 5, wherein said desiccated embryos are frozen following secondary desiccation. 
     
     
       35. A method according to claim 5, wherein the desiccated embryos are rehydrated and germinated in the presence of osmoticum. 
     
     
       36. A method according to claim 5 wherein after step (2) the embryos are coated with a solution comprising a non-hydrated water soluble compound having a melting point ranging between 20 and 70° C., said solution having a temperature slightly above its melting point prior to coating said embryos, thereby allowing said solution to solidify rapidly to yield hardened capsules containing said embryos.